Both genes and environment and determine who will develop asthma. While genome-wide linkage screens and focused analysis of specific candidate loci have made inroads into the question of which genes are culpable in asthma, the preponderance of the genetic effect remains unexplained. A large number of low-frequency codominant mutations are believed to be responsible for most complex genetic diseases, and such mutations are not readily isolated through a genome-wide screen of the population at large. The environmental influence is complex, and in some respects paradoxical. On the other hand, asthma is provoked by environmental allergens. However, in accordance with the Hygiene Hypothesis, heavy exposure to environmental agents that induce an innate immune response can effectively prevent asthma. The physical basis for this effect has come into sharp focus with the finding that ligands such as lipopolysaccharides (LPS) and immunostimulatory oligodeoxyribonucleotides (ISS-ODN), which signal via the Toll-like receptors (TLRs), are able to attenuate airway hyper-reactivity. Hence, a discrete collection of genes has been found to contribute to innate immune sensing, and a practical assay for innate immune sensing has emerged. This proposal is designed to probe the repertoire of genes that link environmental immunostimulants (specifically ISS-ODN) and asthma. We will rely upon N-ethyl-N-nitrosourea (ENU) mutagenesis to identify genes that disrupt signaling initiated by ISS-ODN, and thereby block its ability to prevent airways hyper-reactivity. Mutations are found to have this effect will be isolated through positional cloning, and the genes involved will be characterized to determine the level at which they contribute to signaling. The human orthologs of these genes will be analyzed by DNA sequencing in asthma patients and in ethnically matched controls without asthma. Hence, we will first identify loci wherein isolated mutations clearly producer airways disease in mice. We will then directly measure the effect of genetic load at these loci in humans.